Examination of the presence or absence of cancer cells in a sample collected from a patient can be an indicator for judging cancer metastasis in a tissue or organ from which the sample was collected. For judging the presence or absence of cancer cells in a sample, histological cytodiagnosis such as Papanicolaou staining has conventionally been used. In this method, however, there are problems such as a difference in diagnostic result due to the experience of a person who makes the diagnosis, necessity for a long time in examination, and the like.
Accordingly, studies on molecular pathological diagnosis of cancer by LAMP (loop-mediated isothermal amplification method) and PCR (polymerase chain reaction) have been extensively conducted. Pathological molecular diagnosis can be carried out by detecting a cancer marker gene contained in a tissues and cell (for example, an mRNA of a protein expressed specifically in a cancer cell (hereinafter, also referred to simply as cancer marker)). For example, mRNAs of cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) are known to be effective as cancer marker genes for judging lymph node metastasis of breast cancer. An mRNA of CEA is also known to be effective as a cancer marker for judging metastasis of stomach cancer. These cancer markers are molecules recognized to be significantly different in expression level between a normal sample and a sample containing cancer cells metastasizing to it.
A judgment result of the presence or absence of cancer cells, obtained on the basis of the expression level of a cancer marker, can serve as an indicator for a physician for example to make a diagnosis for metastasis of cancer cells to a specific tissue in a patient.
Conventionally, when such molecular examination is conducted, the expression level of a cancer marker to be analyzed is subjected to conversion (normalization) to the expression level thereof per cell because the number of cells varies from sample to sample. Then, the normalized expression level of the cancer marker in a sample is compared with a threshold value, thereby judging the presence or absence of cancer cells. Specifically, the expression level of a cancer marker gene is divided by the expression level of a housekeeping gene (that is, a gene estimated to be expressed at a constant level in many tissues and cells), thereby normalizing the expression level of the cancer marker gene with the expression level of the housekeeping gene (M. Inokuchi et al., British Journal of Cancer (2003) 89, 1750-1756).
U.S. Publication No. 2007264635 describes a method of obtaining information for prediction of cancer relapse, which comprises amplification reaction of a predetermined gene by PCR and measurement of its amplification product. More specifically, this publication discloses judgment of stomach cancer relapse by normalizing a measurement result with β-actin in Example 4 and judgment of relapse without normalization in Example 5, respectively.